Protein recognition of specific base sequences in double-helical DNA lies at the heart of many biological processes, including the regulation and expression of genetic information and site-specific recombination. The long-term objective of this project is to use restriction endonucleases as models to understand the structural and energetic factors that determine specificity in site-specific DNA-protein interactions. Previous work with EcoRI and BamHl endonucleases has established a rigorous thermodynamic and kinetic basis for a quantitative understanding of the changes in free energy (AG[unreadable]), enthalpy (AH[unreadable]) and entropy (AS0) in formation of protein-DNA complexes. The approach is aimed at uniting structural and energetic perspectives on specificity and at testing the generality of the principles adduced. It is now proposed to determine how molecular strain (DNA bending in the EcoRV complex; electrostatic repulsion in the BamRL complex) affects thermodynamic parameters (AH[unreadable], AS0 and AC[unreadable]p) and to determine how particular sequence contexts (i.e., outside a DNA recognition site) act to modulate specificity and to adjust molecular strain in the DNA complexes of [unreadable]coRI and EcoRV endonucleases. Existing and newly-engineered mutants of EcoRl endonuclease will be used to determine how the introduction of new favorable interactions or the elimination of unfavorable interactions relaxes specificity by modifying the structure of the protein-DNA interface, the thermodynamics of binding and/or the cleavage kinetics of the system. The BamHI and EcoRV endonucleases will be used for quantitative calibration of the excluded-cosolute method of evaluating H2O release during protein-DNA association, and to compare experimental results with computational predictions. PhHhORMANCE SITE(S) (organization, aty, state) Department of Biological Sciences, Univ. of Pittsburgh, Pittsburgh PA 15260 KEY PERSONNEL. See instructions on Page 11. Use continuation pages as neededto provide the required information in the format shown below. Name Organization Role on Project Linda Jen-Jacobson LisaE. Engler Michael R. Kurpiewski DengfengCao Arabela Grigorescu Paul Sapienza John J. Perona John M. Rosenberg Univ. of Pittsburgh Univ. of Pittsburgh Univ. of Pittsburgh Univ. of Pittsburgh Univ. of Pittsburgh Univ. of Pittsburgh Univ. of California, Santa Barbara Univ. of Pittsburgh Principal Investigator Postdoctoral Research Assoc. Research Specialist Graduate Research Assistant Graduate Research Assistant Graduate Research Assistant Collaborator Collaborator PHS398(Rev. 4/98) Patje 2 B B Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3t>. CC Principal Investigator/Program Director (Last, first, middle): Jen-Jacobson. Linda Type the name of the principal investigator/progi^Hlirector at the top of each printed page and each dflbuation page. (Fortype specifications, see instructions on page 6.) ^^ ^^ RESEARCH GRANT TABLE OFCONTENTS Page Numbers Description,